A. Electronic Analysis of Cells
Bioelectronics is a progressing interdisciplinary research field that involves the integration of biomatereials with electronic devices. Bioelectronic methods have been used for analyzing cells and assaying biological molecules and cells. In one type of application, cells are cultured on microelectrodes and cell-electrode impedance is measured and determined to monitor cellular changes.
In PCT Application No. PCT/US03/22557, entitled “IMPEDANCE BASED DEVICES AND METHODS FOR USE IN ASSAYS”, filed on Jul. 18, 2003, a device for detecting cells and/or molecules on an electrode surface is disclosed. The device detects cells and/or molecules through measurement of impedance changes resulting from the attachment or binding of cells and/or molecules to the electrode surfaces. A number of embodiments of the device is disclosed, together with the apparatuses, system for using such devices to perform certain cell based assays.
In PCT Application No. PCT/US04/037696, entitled “REAL TIME ELECTRONIC CELL SENSING SYSTEM AND APPLICATION FOR CELL-BASED ASSAYS”, filed on Nov. 12, 2004, devices, systems and methods for assaying cells using cell-substrate impedance monitoring are disclosed. In one aspect, the disclosed cell-substrate monitoring devices comprise electrode arrays on a nonconducting substrate, in which each of the arrays has an approximately uniform electrode resistance across the entire array. In another aspect, the disclosed cell-substrate monitoring systems comporse one or more cell-substrate devices comprising multiple wells each having an electrode array, an impedance analyzer, a device station that connects arrays of individual wells to the impedance analyzer, and software for controlling the device station and impedance analyzer. In another aspect, the disclosed cellular assays use impedance monitoring to detect changes in cell behavior or state.
In PCT Application No. PCT/US05/004481, entitled “REAL TIME ELECTRONIC CELL SENSING SYSTEM AND APPLICATIONS FOR CYTOTOXICITY PROFILING AND COMPOUND ASSAYS”, filed on Feb. 9, 2005, devices, systems and methods for assaying cells using cell-substarte impedance monitoring are disclosed. In one aspect, the disclosed cellular assays use impedance monitoring to detect changes in cell behavior or state. The methods can be used to test the effects of compounds on cells, such as in cytotoxicity assays. Methods of cytotoxicity profiling of compounds were also provided.
B. Natural Killer Cell-Mediated Cytotoxicity Assays
Natural killer (NK) cells are an integral part of the innate immune response. NK cells have been endowed with the ability to recognize and annihilate cells with extreme stress load such as virus infected cells as well as tumor cells (Lanier, 2005). The death mediated by NK cells is rapid and multifaceted. In addition to their important role in innate immune response, NK cells also serve as key mediators for activation of the adaptive immune response, by secreting factors which serve to activate and propagate B and T lymphocytes (Djeu et al., 2002; Lanier, 2005; Smyth et al., 2005). By responding immediately to infected cells, NK cells keep pathogen infection at bay while giving the adaptive arm of the immune response time to mobilize and respond to pathogen challenge in a more specific manner.
NK cells express specific receptors on its surface that can recognize pathogen infected and tumor cells by their lack of expression or low expression of the major histocompatability complex (MHC) at the membrane (Cerwenka and Lanier, 2001; Lanier, 2005). These receptors can be both inhibitory and stimulatory and the combined action of these receptors determines the extent of the dual nature of NK cell response to target cells; namely cytotoxicity and/or cytokine production (Lanier, 2005). When NK inhibitory receptors bind to MHC class I molecules, their effector functions are blocked and therefore normal healthy cells which express adequate levels of these receptors are spared from the NK cell attack. On the other hand activating receptors such as NKG2D is engaged by ligands that are MHC-like and expressed by pathogen infected and transformed cells as well other stressed cells (Cerwenka and Lanier, 2001). The immediate effector function of NK cells results in the release of secretory granules mainly containing special proteins and enzymes of the perforin family and granzyme family (Smyth et al., 2005). The perforins bind to the cell membrane and disrupt the integrity of the plasma membrane whereas granzymes are a special class of serine proteases with various substrate specificities, including caspases (Trapani and Smyth, 2002). The combined interplay of these proteins and enzymes ultimately result in target cell cytotoxicity and destruction. In addition to granzyme and perforin-mediated cytotoxicity, NK cells also express Trail and FasL, both of which can contribute to NK-mediated cytotoxicity towards target cells (Smyth et al., 2002).
A number of techniques have been devised for monitoring NK-mediated cytotoxicity towards target cells. The most popular method relies on chromium 51 labeling of target cells and measuring the release of chromium upon cytolysis (Brunner et al., 1968). Other label-based methods such as annexin-V staining of target cells and using fax analysis to analyze apoptotic cells have also been described (Goldberg et al., 1999). Enzymatic assays which measure the activity of certain enzymes such as lactate dehydrogenase (LDH) or Granzymes have also been described in the literature as a means to assess NK-mediated cytotoxicity (Korzeniewski and Callewaert, 1983; Ewen et al., 2003; Shafer-Weaver et al., 2003). While all of the above mentioned assays, especially chromium 51 release assay have proved informative and are in routine use, they all have certain drawbacks which limit their utility. For example, chromium labeling involves the usage of a radioactive label which can be hazardous and difficult to dispose of. Furthermore, chromium 51 assay window is limited to four hours beyond which the natural tendency of chromium to diffuse out of the cell contributes to high background. In addition, all the mentioned assays are end-point assays which provide a “snapshot” of the NK-mediated cytotoxicity activity and with respect to chromium 51 release assay, it would be difficult to measure NK-mediated target cell killing after the 4 hour time window. Thus, technologies or methods that do not the use of radioactive labels and can provide kinetic information about NK-mediated cytotoxicity process are needed.
The present invention further expands the inventions disclosed in PCT Application No. PCT/US03/22557, entitled “IMPEDANCE BASED DEVICES AND METHODS FOR USE IN ASSAYS”, filed on Jul. 18, 2003, and disclosed in U.S. patent application Ser. No. 10/705,447, entitled “IMPEDANCE BASED DEVICES AND METHODS FOR USE IN ASSAYS,” filed on Nov. 10, 2003, and disclosed in PCT Application No. PCT/US05/004481, entitled “REAL TIME ELECTRONIC CELL SENSING SYSTEM AND APPLICATIONS FOR CYTOTOXICITY PROFILING AND COMPOUND ASSAYS”, filed on Feb. 9, 2005, and disclosed in U.S. patent application Ser. No. 11/055,639, entitled “REAL TIME ELECTRONIC CELL SENSING SYSTEM AND APPLICATIONS FOR CYTOTOXICITY PROFILING AND COMPOUND ASSAYS” filed on Feb. 9, 2005, and disclosed in PCT Application No. PCT/US04/037696, entitled “REAL TIME ELECTRONIC CELL SENSING SYSTEM AND APPLICATION FOR CELL-BASED ASSAYS”, filed on Nov. 12, 2004, and disclosed in U.S. patent application Ser. No. 10/987,732, entitled “REAL TIME ELECTRONIC CELL SENSING SYSTEM AND APPLICATION FOR CELL-BASED ASSAYS” filed on Nov. 12, 2004. The present invention provides a real time cell electronic sensing system for conducting cell-based assays based on measurement of cell-substrate impedance and provides the method for using such a system for assaying for natural killer, cytotoxic T-lymphocyte and neutrophil-mediated killing of target cells using real-time microelectronic cell sensing technology.